Paralytic Shellfish Poisoning (PSP) has been known for centuries and it has been responsible for many deaths (Kao, 1966). The toxins responsible of PSP are tetrahydropurines that block sodium channels, resulting in respiratory and heart paralysis (Hall, 1982). At least 18 types of PSP toxins have been described (FIG. 1), mainly from marine dinoflagellates and shellfish that feed on toxic algae. Attempts to isolate PSP toxins began more than one century ago (Salkowski, 1885), but their occurrence as mixtures of compounds with diverse ionizable residues complicated their purification. The development of ionic exchange chromatography, guided by mouse bioassay, eventually allowed the isolation of a basic toxin, water soluble, from Alaska clams (Saxidomas giganteus) (Schantz et al., 1957). This compound was named saxitoxin (STX) and therefore the group of paralytic toxins, saxitoxins (Schuett and Rapoport, 1962). The STX structure is shown in FIG. 1 and was established by X ray crystalography (Schantz et al., 1975) and chemical synthesis (Tan et al., 1977; Kishi, 1980; Jacobi et al., 1984; Martinelli et al., 1986).
In most cases, PSP toxins correspond to sulphatated derivatives of STX, such as the 11-hydroxysaxitoxin sulphates (gonyautoxins GTX2 and GTX3) or N-sulphocarbamoyl derivatives (B1, C1 and C2). It is possible to find also the N-1-hydroxysaxitoxin or neosaxitoxin (NEO) and their sulphates (B2, GTX1, GTX4, C3 and C4), as well as the less common decarbamoyl toxins (FIG. 1) (Sullivan et al., 1983). The STXs potencies, measured by mouse bioassay, vary enormously. Generally, the carbamoyl toxins are the most potent, the sulphocarbamoyl toxins are the less potent, and the decarbamoyl toxins have intermediate potency (Oshima et al., 1992).
Shellfish acquire and concentrate the STXs as a result of feeding with toxic dinoflagellates. Several species of dinoflagellates have been associated with paralytic toxins, including Alexandrium catenella (Schantz et al., 1966; Proktor et al., 1975; Bates et al., 1978), A. excavatum (Desbiens et al., 1990), A. fundyense (Anderson et al., 1990) and A. tamarensis (Prakash, 1967; Anderson and Po-on Cheng, 1988) in the northern latitudes, and in the southern latitudes, Gymnodinium catenatum, Pyrodinium bahamense (Taylor, 1985; Anderson et al., 1989) and Gonyaulax polyedra (Bruno et al., 1990). The dinoflagellate cysts, deposited in marine sediments, can remain toxic for several months (Selvin et al., 1984). The composition of paralytic toxins varies enormously depending on the dinoflagellate specie from which they were isolated (Boyer et al., 1985; Cembella et al., 1987). Also there are intra-specie variations (Maranda et al., 1985; Cembella et al., 1987). However, toxin composition of a certain dinoflagellate strain, isolated from a particular geographical zone, is extremely constant.
Shellfish produces important changes in the paralytic toxin profile. Due to the differences in toxin potencies, a shellfish can change drastically its total toxicity without modifying the total quantity of toxin (Oshima et al., 1990). Other changes in the toxin profile can occur due to non enzymatic processes. Without exception, the gonyautoxins suffer epimerization, with the equilibrium displaced to the alpha forms, that are energetically more favourable (Fix Wichmann et al., 1981; Hall, 1982). The conversion speed is dependant on the pH and chemical structure, with a faster epimerization near to neutral pH. All paralytic toxins are quickly oxidized to non toxic products if the pH is not controlled during their extraction. Conditions of neutral and alkaline pH favour the oxidation. Under extremely acidic pHs (1M of free acid) carbamoyl groups are removed, while at pH 1 and 100.degree. C. the lost group corresponds to the sulphate (sulphocarbamoyl) of the sulphocarbamoyl toxins, with a complete conversion in 5 min (Hall and Reichardt, 1984). Due to the low toxicity of the sulphocarbamoyl toxins and to the high toxicity of the carbamoyl toxins, the loss of the sulphate group produces an increase in the total toxicity.
Although it is possible to predict the time of the year, and in some parts of the world, the exact localization of the PSP proliferation, the toxicity vary enormously from year to year. Therefore, monitoring programs are absolutely necessary in order to protect the shellfish industry and the consumer. The mouse bioassay has been a standard method for PSP toxins detection and quantification for more than 50 years (Sommer and Meyer, 1937; McFarren, 1958; Helrich, 1990). Due to the use of experimental animals, the variability of the results and because the sensitivity of the mouse bioassay is very close to the regulatory limits, attempts have been made to replace this method with other methods, for example, toxin detection by HPLC. However, the mouse bioassay is simple and quick. On the other hand, this method is a direct measurement of toxicity, which is an important consideration for the security of the shellfish, particularly because of the discovery of new toxins. The HPLC method is based on a chromatographic ion pairing-separation of toxins in a RP8 column. Subsequently, through a post column derivatization, toxins are alkaline oxidated and then fluorometrically detected (Sullivan et al., 1988; Oshima et al., 1988).
The occurrence of intoxication due to paralytic toxin has increased consistently throughout the world in the past years. Until 1970, some 1700 cases of PSP have been registered, mainly in North America and Europe (Prakash et al., 1971). On the other hand, in the period 1971-1984 around 900 additional cases have been described, especially in zones of the world where PSP was practically unknown (WHO 1984). In China, PSP appeared at the beginning of the 1950s, in Japan and Norway at the end of the 1960s, in Malaysia, The Philippines, New Guinea, Australia, Indonesia, Argentina and Chile at the end of the 1970s and the beginning of the 1980s, in Sweden, Denmark, Guatemala, Venezuela, Mexico and Uruguay at the end of the 1980s and the beginning of the 1990s. Actually large parts of the world-wide coasts have or had recurrent proliferation of the PSP producer algae. This has generated a growing problem in Public Health, especially concerning the prevention of intoxication in human beings. With the continual increase in the world-wide areas that are contaminated with PSP producer algae, the areas designated for shellfish collection and cultivation are becoming more limited. This has caused a reduction in production in many areas, with a consequent socioeconomic impact of the involved fishermen. This situation is driving the need to evaluate seriously how to utilize PSP contaminated shellfish.
One alternative is to use PSP-containing shellfish detoxified to levels that are not toxic to human beings. The maximal level of PSP accepted on a world-wide basis as being safe for shellfish was 80 .mu.g of equivalent STX for each 100 g of mollusc flesh
Shellfish PSP detoxification data found in the literature are scarce and non systematic. The proposed shellfish detoxification processes can be grouped in 4 classes of strategies:
1.--Detoxification of live shellfish. PA1 2.--Chemical treatment. PA1 3.--Removal of the more toxic parts. PA1 4.--Processing.
1.--Detoxification of live shellfish
This can be done by transplanting the toxic shellfish to a non toxic area. In this circumstance the shellfish suffer detoxification by depuration unless re-toxification occurs. Actually detailed information is known only about the detoxification kinetic of 3 species of scallops: Patinopecten yessoensis, Placopecten magellanicus, and Chlamys nipponensis (Medcof et al., 1947; Jamieson and Chandler, 1983; Shumway et al., 1988). The facts now available suggest that within the filter-feeding bivalve molluscs, scallops can be classified as species that retain the toxins for a long time. For example, the retention of paralytic toxins in P. magellanicus has been identified for periods that run from several months to 2 years (Medcof et al., 1947; Jamieson and Chandler, 1983; Shumway et al., 1988). Certain tissues of P. magellanicus, particularly the digestive glands and rims, can remain toxic through a year (Bourne, 1965; Shumway et al., 1988). Very similar results were observed for the pink scallop Chlamys hastata (Nishitani and Chew, 1988).
Despite the fact that this method is technically feasible, the cost to transfer, transplant and re-seed makes this decontamination procedure economically impracticable.
2.--Chemical treatment
Knowing the paralytic toxin formulas and together with some empirical observations, Hayes (1966) proposed that toxins could be extracted and/or destroyed under acidic conditions, particularly in the presence of oxygen. Based on this observation, experiments were carried out in Alaska with live and dead clams and clam flesh under acidic conditions. It was observed that at a pH as low as 5.0, a reduction of the toxicity levels was not observed (Hayes, 1966).
3.--Removal of the more toxic parts
The PSP toxins distribution in bivalve shellfish tissues are variable and it has been demonstrated that the distribution depends on the species involved. The most studied shellfish are scallops and clams. In FIG. 2 the most important anatomical parts of scallops and clams are depicted.
In general, it has been determined that the digestive glands (hepatopancreas and liver) usually possess the highest levels of paralytic toxins. Rims (mantels, rings, or borders), gills and gonads (roe) also possess significant quantities of paralytic toxins, although levels lower than those from the digestive glands. Finally, adductor muscles and the foot are tissues that always, except in a very few instances, possess very low levels of toxins (see Table 1) (Shumway and Cembella, 1993; Cembella et al., 1993; Cembella and Shumway, 1995).
TABLE 1 Anatomical distribution of paralytic toxins in scallop and clam. Relative contribution to the Relative toxicity Mullusc Tissue total weight (%) (% of total toxicity) Scallop Digestive gland 21 69-75 Rim 11 23-27 Gonad 23 1-2 Gill 10 1-2 Adductor Muscle 36 &lt;1 Clam Digestive gland 18 37 Rim 14 20 Siphons 10 13 Gill 6 21 Foot 30 7 Adductor muscle 22 2 *Data obtained from Shumway and Cembella, 1993; Cembella et al., 1993; Cembella and Shumway, 1995.
One of the clearest examples of the utilization of this "decontamination" procedure applies to scallops. In North America, the effects of algal blooms on scallop cultivations are ignored, since only the adductor muscle is consumed. As indicated in table 1, the adductor muscle is frequently the tissue with the lowest level of paralytic toxins. Once scallops are harvested, almost immediately the adductor muscle is removed and the rest of the tissues together with the shells, are discarded. The discarded tissues, and those include rim (mantel, rings or borders), gonad (roe), digestive gland (hepatopancreas, liver) and gills, correspond to approximately 65% of the total mollusc weight (Cembella et al., 1993).
However, in some markets, such as Latin America, Europe and Australia, scallops are sold together with the gonad, or whole. In this case, as well as in many other cases, where molluscs are sold, this procedure is not applicable.
4.--Processing
4.1.--Cooking and canning
It have been described that just the cooking of contaminated molluscs can reduce their toxicity level. Initial work with paralytic toxins demonstrated that the heat could destroy an important portion of the toxins. Medcof et al. (1947) and Quayle (1969) described the reduction of total toxicity of toxic fresh molluscs when these were subjected to home cooking processes, like boiling or frying. They also demonstrated, using a clam (Mya arenaria), that commercial canning was more effective than domestic cooking in reducing the PSP toxicity. It was demonstrated that pre-cooking with water vapour for 10 min, of low PSP toxicity shellfish, succeeded in reducing their toxic levels in approximately 90%, but posterior treatments with water vapour didn't succeed in further reducing toxicity. A latter treatment in an autoclave at 121.degree. C. (250.degree. F.) for 45 min reduced the toxic content only an additional 3%, and 90 min in an autoclave caused an additional reduction of 1%. Quayle (1969), Prakash et al. (1971), Noguchi et al. (1980) and Berenguer et al. (1993) have contributed important observations concerning toxicity reduction during commercial canning process. Recently, it has also been reported that the speed of thermal degradation of some PSP toxins depends on the type of toxin involved and on the temperature (Nagashima et al., 1991).
Table 2 summarized some data obtained on toxicity reduction by commercial canning. In general, it was observed that the commercial canning of molluscs that possess an initial toxicity greater than 1000 mg STX eq./100 g achieves a significant decontamination level, but their final toxicity is not adequate for human consumption. This detoxification procedure by canning would only serve for the decontamination, with certain level of security, of molluscs that possess up to 500 .mu.g STX eq./100 g.
4.2.--Freezing
Short freezing times do not reduce substantially the PSP toxin levels. Only after several months of storage at -20.degree. C. were small reductions in toxicity detected. However, the freezing of whole molluscs results in a migration of toxins from more to fewer toxic parts, for example, from digestive glands to adductor muscles. This phenomenon is also achieved during the thawing of the molluscs (Shumway and Cembella, 1993).
TABLE 2 PSP toxicity (.mu.g of saxitoxin equivalent per 100 g of mollusc) in raw material and in final canned product. Toxicity (.mu.g STXeq./100 g)* Raw material Canned product Mollusc toxicity toxicity ** Reference Mya arenaria 5,000-6,000 310 @ Medcof et al., 1947 1,300 200 @ 1,000-1,100 &lt;200 500-1,000 &lt;200 250-500 &lt;200 200-250 &lt;200 700-800 &lt;200 5,000-5,800 310 @ Saxidomus 192 32 Quayle, 1969 giganteus 48 32 126-176 32 240 32 192-352 32 368-560 32 787 102 @ 66 32 78 32 144 32 149 32 284 32 1,126 81 @ 415 109 @ Mya arenaria 112-138 &lt;32 Prakash et al., 1971 800-930 50 800-960 50 210 32 160-175 &lt;32 80-160 &lt;32 40-80 &lt;32 32-40 &lt;32 Acanthocardia 799 &lt;35 Berenguer et al., 1993 tuberculatum L. 803 &lt;35 269 &lt;35 428 &lt;35 * Toxicity level was determined by mouse bioassay. ** All molluscs were canned as wholes. @ Molluscs not for human consumption.